GLUCOAMYLASE VARIANTS AND POLYNUCLEOTIDES ENCODING SAME

• US 20200131491A1
• Filed: 04/10/2018
• Published: 04/30/2020
• Est. Priority Date: 04/11/2017
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Namoto, Tomoko, Tsutsumi, Noriko, Ayabe, Keiichi, Kang, Zhengfang
• Granted Patent: US 11,279,921 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to glucoamylase variants having an increase in raw starch activity compared to the guy-coamylase disclosed as SEQ ID NO: 2, comprising one or more modifications in the catalytic domain and/or one or more modifications in the starch binding domain selected from: a) at least one, preferably at least two, preferably at least three, preferably at least four of: V18M, T43K, N112L, T116R, A117Q, G120S, A271F, Y295W, Q318Y; and/or b) Introducing at least three, preferably at least four substitutions selected from the group: S458C, S458SCGG, S458SGGC, A493V, A518K, E520Q, N527M, S540K, R, S(G)546P, T(V)549W, N503R, N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Claims

1. A glucoamylase variant, having increased raw starch hydrolytic activity at pH=4.0, T=32°

C., compared to the glucoamylase disclosed in SEQ ID NO;

2, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO;

2 or 1-454 of SEQ ID NO;

4, a linker comprising amino acids 455-462 of SEQ ID NO;

2 or amino acids 455-465 of SEQ ID NO;

4 and a starch binding domain comprising amino acids 463-556 of SEQ ID NO;

2 or amino acids 466-559 of SEQ ID NO;

4, and wherein the variant further comprises a substitution at one or more positions selected from the group consisting of;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R;

V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+Q318Y+S95P+A121P+Y295W+S458SCGG+N527M+T(V)549W;

S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+A518K+N527M+T549W;

T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+N527M+T549W;

S95P+A121P+Y295W+T116R+N527M+T(V)549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+A117Q+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W;

T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W;

S95P+G120S+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+A493V+A518K+E520Q+T549W;

S95P+A121P+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A518K+T(V)549W;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458C+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+N527M+T549W;

S95P+A121P+A271F+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A493V;

S95P+A121P+Y295W+S540K;

S95P+A121P+Y295W+S540R;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458SGGC+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+N527M+T(V)549W; and

S95P+A121P+Y295W+S458SCGG+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

wherein the position numbering corresponds to amino acid positions in the amino acid sequence set forth in SEQ ID NO;

2; and

wherein the variants have at least 85%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO;

2.

2. The glucoamylase variant of claim 1, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO:

2, a linker comprising amino acids 455-465 of SEQ ID NO;

4 and a starch binding domain comprising amino acids 466-559 of SEQ ID NO;

4, and wherein the variant comprises a combination of specific substitutions selected from the group consisting of;

V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R;

V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+S458SCGG+N527M+T(V)549W;

S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R; and

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

and wherein the increase in raw starch hydrolytic activity compared to SEQ ID NO;

2 is at least 30%.

3. The glucoamylase variant of claim 1, wherein the variant is derived from the glucoamylase of SEQ ID NO:

2, and wherein the variant comprises a combination of specific substitutions selected from the group consisting of;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+A121P+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W;

T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+A493V+A518K+E520Q+T549W;

S95P+A121P+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+N527M+T549W;

S95P+A121P+A271F+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A493V;

S95P+A121P+Y295W+S540K;

S95P+A121P+Y295W+S540R; and

S95P+A121P+Y295W+A518K+N527M;

and wherein the increase in raw starch hydrolytic activity compared to SEQ ID NO;

2 is at least 5%.

4. The glucoamylase variant of claim 1, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO:

2, a linker comprising amino acids 455-465 of SEQ ID NO;

4 and a starch binding domain comprising amino acids 466-559 of SEQ ID NO;

4, and wherein the variant comprises a substitution at one or more positions selected from the group consisting of;

T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

S95P+A121P+Y295W+T116R+N527M+T(V)549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+A117Q+N527M+T(V)549W;

S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W;

S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+A518K+T(V)549W;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458C+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+S458SGGC+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+N527M+T(V)549W; and

S95P+A121P+Y295W+S458SCGG+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

and wherein the increase in raw starch hydrolytic activity compared to SEQ ID NO;

2 is at least 2%.

5. The glucoamylase variant of claim 1, wherein the number of substitutions or insertions are 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions or insertions.

8. A composition comprising the glucoamylase variant of claim 1.

10. A process of producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying starch-containing material in the presence of an alpha amylase;

(b) saccharifying the liquefied material; and

(c) fermenting with a fermenting organism;

wherein step (b) is carried out using at least a glucoamylase variant of claim 1.

11. The process of claim 10, wherein step (b) and step (c) are carried out simultaneously.

12. A process of producing a fermentation product from starch-containing material, comprising the steps of:

(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and

(b) fermenting with a fermenting organism,

wherein step (a) is carried out using at least a glucoamylase variant of claim 1.

13. A process of producing a syrup product from starch-containing material, comprising the step of:

(a) liquefying starch-containing material in the presence of an alpha amylase;

(b) saccharifying the liquefied material in the presence of a glucoamylase variant of claim 1.

14. The process of claim 10, wherein the fermenting organism is selected from a Saccharomyces spp. yeast.

15. The process of claim 14, wherein the yeast expresses a glucoamylase having at least 90% identity to SEQ ID NO:

2 or SEQ ID NO;

4.

16. A polynucleotide encoding the glucoamylase variant of claim 1.

17. A nucleic acid construct comprising the polynucleotide of claim 16.

18. An expression vector comprising the polynucleotide of claim 16.

19. A host cell comprising the polynucleotide of claim 16.

20. The process of claim 10, wherein a host cell is applied in the fermentation step, wherein the host cell comprises a polynucleotide encoding a glucoamylase variant having increased raw starch hydrolytic activity at pH=4.0, T=32°

C., compared to the glucoamylase disclosed in SEQ ID NO;

2, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO;

2 or 1-454 of SEQ ID NO;

4, a linker comprising amino acids 455-462 of SEQ ID NO;

2 or amino acids 455-465 of SEQ ID NO;

4 and a starch binding domain comprising amino acids 463-556 of SEQ ID NO;

2 or amino acids 466-559 of SEQ ID NO;

4, and wherein the variant further comprises a substitution at one or more positions selected from the group consisting of;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R;

V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+Q318Y+S95P+A121P+Y295W+S458SCGG+N527M+T(V)549W;

S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+A518K+N527M+T549W;

T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+N527M+T549W;

S95P+A121P+Y295W+T116R+N527M+T(V)549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+A117Q+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W;

T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W;

S95P+G120S+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+A493V+A518K+E520Q+T549W;

S95P+A121P+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A518K+T(V)549W;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458C+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+N527M+T549W;

S95P+A121P+A271F+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A493V;

S95P+A121P+Y295W+S540K;

S95P+A121P+Y295W+S540R;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458SGGC+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+N527M+T(V)549W; and

S95P+A121P+Y295W+S458SCGG+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

wherein the position numbering corresponds to amino acid positions in the amino acid sequence set forth in SEQ ID NO;

2; and

wherein the variants have at least 85%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO;

2.

21. A method of producing a glucoamylase variant, comprising:

(a) cultivating the host cell of claim 19 under conditions suitable for expression of the variant; and

(b) optionally recovering the variant.

22. The process of claim 12, wherein a host cell is applied in the fermentation step, wherein the host cell comprises a polynucleotide encoding a glucoamylase variant having increased raw starch hydrolytic activity at pH=4.0, T=32°

C., compared to the glucoamylase disclosed in SEQ ID NO;

2, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO;

2 or 1-454 of SEQ ID NO;

4, a linker comprising amino acids 455-462 of SEQ ID NO;

2 or amino acids 455-465 of SEQ ID NO;

4 and a starch binding domain comprising amino acids 463-556 of SEQ ID NO;

2 or amino acids 466-559 of SEQ ID NO;

4, and wherein the variant further comprises a substitution at one or more positions selected from the group consisting of;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R;

V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+Q318Y+S95P+A121P+Y295W+S458SCGG+N527M+T(V)549W;

S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R;

S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+A518K+N527M+T549W;

T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

T43K+S95P+A121P+Q318Y+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+N527M+T549W;

S95P+A121P+Y295W+T116R+N527M+T(V)549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+A117Q+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;

T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W;

T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;

S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W;

T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W;

V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;

S95P+G120S+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+N527M+T(V)549W;

S95P+A121P+Y295W+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;

T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W;

S95P+G120S+A121P+Y295W+A518K+N527M+T(V)549W;

S95P+N112L+A121P+Y295W+A518K+N527M+T(V)549W;

V18M+T43K+S95P+A121P+Q318Y+A518K+T549W;

S95P+A121P+Y295W+A493V+A518K+E520Q+T549W;

S95P+A121P+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A518K+T(V)549W;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458C+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

S95P+A121P+Y295W+N527M+T549W;

S95P+A121P+A271F+Y295W+A518K+N527M+T549W;

S95P+A121P+Y295W+A493V;

S95P+A121P+Y295W+S540K;

S95P+A121P+Y295W+S540R;

S95P+A121P+Y295W+A518K+N527M;

S95P+A121P+Y295W+S458SGGC+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

T43K+S95P+A121P+Y295W+N527M+T(V)549W; and

S95P+A121P+Y295W+S458SCGG+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

wherein the position numbering corresponds to amino acid positions in the amino acid sequence set forth in SEQ ID NO;

2; and

wherein the variants have at least 85%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO;

2.

6. A method for increasing raw starch hydrolysis activity of a glucoamylase comprising the steps:

(a) providing a hybrid glucoamylase comprising a first amino acid sequence consisting of amino acids 1-454 of SEQ ID NO;

2, a second amino acid sequence selected from amino acids 455-462 of SEQ ID NO;

2 or amino acids 455-465 of SEQ ID NO;

4, and a third amino acid sequence selected from amino acids 463-556 of SEQ ID NO;

2 or amino acids 466-559 of SEQ ID NO;

4; and

/or

(b) introducing a substitution selected from the group consisting of;

V18M, T43K, N112L, T116R, A117Q, G120S, A271F, Y295W, Q318Y; and

/or

(c) Introducing at least three substitutions selected from the group consisting of;

S458C, S458SCGG, S458SGGC, A493V, A518K, E520Q, N527M, S540K, R, S(G)546P, T(V)549W, N503R, N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A.

7. A method for increasing raw starch hydrolysis activity of a glucoamylase comprising the steps:

(a) providing a hybrid glucoamylase comprising a first amino acid sequence consisting of amino acids 1-454 of SEQ ID NO;

2, a second amino acid sequence selected from amino acids 455-465 of SEQ ID NO;

4, and a third amino acid sequence selected from amino acids 466-559 of SEQ ID NO;

4; and

/or

(b) introducing a substitution selected from the group consisting of;

V18M, T43K, T116R, G120S, Y295W, Q318Y; and

/or

(c) introducing a combination of alterations selected from the group consisting of;

i) N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;

ii) N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;

iii) at least three substitutions selected from the group;

S458SCGG, N527M, T(V)549W, and N503R.

9. (canceled)